Bio-processing is important in chemicals and drugs production. In many cases, when conventional chemical syntheses are not feasible or economical, bioprocessing is the only option for use. In general, bio-processes require gentler processing conditions and pose lesser environmental pollution problems than chemical processes do. However, a bioprocess usually has slower reaction rates than its chemical counterpart, and thus is often not chosen for use due to economic considerations.
The bioreaetor is the center-piece of a bioprocess. The conventional bioreactors are derived from the reactors originally designed for homogeneous, chemical reactions. Most of industrial bioreactors in use today are not designed or operated optimally for multiphase, heterogeneous bio-reactions. Some major reasons that most bioprocesses have low reaction rates include the low (active) cell density in the bioreactor and the strong inhibition caused by the reaction products.
Cell immobilization in a bioreactor has long proved to be an effective method to improve reactor productivity of fermentation processes. It facilitates the separation of cells from products in solution and allows reactor operation at high dilution rates without cell washout. However, there are some major problems associated with conventional immobilized cell bioreactors that have prevented their wide industrial applications.
In general, cell immobilization is achieved either by cell entrapment within a confined volume through the use of a polymeric matrix or membrane, or by cell attachment via adsorption or covalent binding to a fixed surface, such as in a biofilm reactor. In conventional cell entrapment systems, overgrowth of cell biomass often causes serious problems during long-term operation of the bioreactor. Diffusion limitations and accumulation of dead cells over time result in loss of cell viability and thus reactor productivity. Also, conventional cell entrapment systems are not suitable for bio-processes which produce or use gases.
For a non-growing, immobilized cell system, the loss of cell viability and thus reactor productivity over time severely limits the operating life of the bioreactor. For an actively growing system, complications in maintaining bioreactor stability make continuous operation of the bioreactor rather difficult at industrial scale. For example, packed-bed and membrane, including hollow-fiber, bioreactors tend to get clogged quickly by cell biomass and fluidized-bed bioreactors are subject to unstable bed expansion due to biofilm growth. Conventional packedbed and membrane bioreactors also suffer from high pressure drop and gas entrapment inside the reactor bed that reduce the reactor working volume substantially. Nutrients transport in these immobilized cell systems also may become a problem due to diffusion limitations. Furthermore, conventional packed-bed and membrane bioreactors tend to accumulate aged or dead cells and gradually lose their production capability over time.
Product inhibition is another major factor in limiting bioprocess productivity. The product from a fermentation process usually is also an inhibitor to the cells used in the process. The removal of the fermentation product from the bioreactor thus can alleviate the product inhibition problem and improve reactor productivity by severalfold. In operation, the process integrates the downstream separation process with the fermentation, and is referred to as extractive fermentation. The extractive fermentation not only improves bioreactor productivity, it also makes the downstream product purification easier. Also, in some cases, extractive fermentation gives higher product yields than the conventional processes. An extractive fermentation involves the use of a second phase which is immiscible with the aqueous phase (fermentation broth), where bioreactions occurs, to continuously remove the product from the aqueous phase. The second phase used can be an organic solvent, polymeric aqueous solution, or a gas.
A packed-bed, immobilized cell structure has now been developed that overcomes the aforementioned problems. This bioreactor has been successfully used in long-term continuous fermentations for biochemicals production. In this new bioreactor, cells are immobilized within a convoluted fibrous matrix packed in a container. The convoluted fibrous structure is illustrated in FIG. 1. In this structure, the fibrous matrix provides large surface areas for cell attachment and a large void space for cell entrapment. Mass transfer limitations within the fibrous matrix can be controlled by using a proper thickness of the matrix layer. Growth of cells to a high density (40.about.100 g/L) thus can occur within the fibrous matrix. Also, the built-in vertical gaps among the spiral-wound layers of the fibrous matrix allow excess cell biomass to fall off to the bottom of the reactor, gases such as CO.sub.2 and air to flow upward freely and escape from the top of the reactor, and the liquid medium to be pumped through the reactor bed without substantial pressure drop. Furthermore, the binding (adsorption) of cells to fiber surfaces can be regulated by the surface properties of the fiber. For example, loose cell attachments to hydrophilic fiber surfaces (such as cotton) would provide renewable surfaces for new cells and prevent aging or degeneration problems. The fibrous matrix functions as filter media and helps to retain cells (but not permanently) in the bioreactor. There is continual growth of new cells and sloughing-off of aged cells in the reactor. Therefore, the bioreactor is able to operate continuously for long periods without observable loss in its productivity. These operating advantages cannot be easily realized using conventional immobilized cell systems.
The new bioreactor can be effectively used in both aerobic and anaerobic processes. It also can be operated either as liquid-continuous or gas-continuous (trickle bed). In the trickle bed configuration, the gas (air) stream flows upward mainly through the spaces between the spiral matrix layers, while the liquid (water) stream flows downward through the fibrous matrix. The highly porous fibrous matrix provides high specific surface areas for cell attachment and for gas-liquid-solid contacts. Also, the large void space (&gt;90%) within the fibrous matrix allows a large reactor working (liquid) volume for cell growth and reactions to take place. The fibrous bioreactor also can be used in extractive fermentation. The medium phase will be passed through the fibrous matrix, while the extractant phase through the gap between fiber layers. This novel bioreactor thus is versatile for use in various bioprocesses with multiphase flows and will find important applications in fermentation, biotransformation and bioffitration.
This new bioreactor has been tested in laboratory studies for several fermentation and extractive fermentation processes, including ethanol and recombinant protein production with yeasts, and organic acids (lactate, acetate and propionate) production with bacterial cultures. In all cases, superior reactor performance (e.g., three to tenfold increases in productivity and up to 1 year stable continuous operation) was obtained. It is reasonable to anticipate that this new bioreactor also will have advantageous applications in other bioprocesses such as in waste water treatment, bioffitration, biotransformation, and cell cultures.
Recently, extractive recovery of carboxylic acids from dilute, aqueous solutions such as fermentation broth and wastewater, which have acid concentrations lower than 10% (wt/wt), has received increasing attention. The extraction of organic acids using long-chain, aliphatic amines is especially important to the recovery and purification of organic acids or their salts from fermentation broth. For example, the acetate produced from a homoacetogenic fermentation is a strong inhibitor to the homoacetogen. Consequently, the fermentation rate would decrease dramatically as acetate is being produced. Also, the acetate concentration in the fermentation broth rarely reaches 4% (wt/vol) to allow economical recovery of acetate using conventional solvent extraction or distillation methods. A new two-step extractive separation of organic acids, such as acetate, by using aliphatic amines is developed (see FIG. 2) to overcome these problems. In this two-step extraction, the organic acid, such as acetic acid, present in the broth is first extracted with the extractant, such as Alamine 336, under acidic conditions. The extractant containing the organic acid is then back-washed or stripped with a concentrated alkaline solution to regenerate the extractant and to form the organic salt in concentrated solution simultaneously. The result is a concentrated organic salt solution that can be further concentrated or dried directly to form the final product. This method significantly cuts the energy costs in recovering and purifying organic acids or salts from dilute aqueous solutions. Also, an extractive fermentation which integrates the fermentation and extraction, as shown in FIG. 2, can be used to remove the fermentation product, such as acetic acid, from the bioreactor during fermentation and thus to reduce product inhibition and to enhance reactor productivity.
A particularly advantageous application of the structure and method of the present invention has been found in the conversion of fermentable sugars into organic acids and the salts of such acids. For example, lactose may be converted to lactic acid, acetic acid, propionic acid or the salts of these organic acids with appropriate fermentation cultures. All such conversions may be effected by known culturing means, however the use of the apparatus and method of the present invention substantially enhances the effectiveness of such conversions. Applications of the present inventions to convert the lactose content of whey into commercially useful calcium magnesium acetate, potassium acetate, calcium propionate, and sodium lactate have been found to be particularly advantageous.
Whey is a byproduct from the manufacture of cheese and casein. It contains about 5% lactose, 1% protein, 1% salts, and 0.1-0.8% lactic acid. The BOD (biological oxygen demand) content of whey is high--40,000 mg/L. The annual production of cheese whey in the United States has continuously increased to about 57 billion pounds (26 million metric tons) in 1988. Currently, only about 50% of the whey produced in the United States is used in human food and animal feed. The rest must find a new use or be treated as pollutant because of the high BOD content of whey. With continuous increases in milk and cheese production in the United States and throughout the world, the disposal of surplus cheese whey is one of the most critical problems facing the dairy industry.
While whey protein generally can be recovered from whey via ultrafiltration, the remaining lactose stream (whey permeate) represents a major disposal problem. Lactose accounts for 70%-80% of total whey solids. It can be readily isolated and purified from whey permeate by crystallization. However, the U.S. and world markets for lactose are cyclical and often very competitive. The market prices for lactose have fluctuated between $0.10/lb and $0.40/lb in the recent past. Furthermore, the lactose recovery yield from whey permeate is low (only about 60%), and the waste stream (commonly called mother liquor or de-lactosed whey permeate) from the crystallization process contains high salts (&gt;20%), high lactose (.about.20%) and high BOD. Because of the high salt content, this mother liquor has limited applications and generally requires costly disposal. The increasing disposal costs have prompted continuous searches for better uses of whey, whey permeate, and de-lactosed whey permeate.
The utilization of whey lactose as a fermentation feedstock has been of great interest to the dairy industry. A wide range of products can be obtained from whey fermentations, including single cell protein, methane, alcohols (ethanol, butanol), organic acids (lactic, acetic, propionic, citric), vitamins, and biopolymers (xanthan gum etc.). However, production of a suitable fermentation product from whey must take into account technological, market, and economic factors. None of the existing whey fermentation processes have achieved wide-scale use in the dairy industry.
Most organic acids are presently produced via petrochemical routes due to the poor reaction rate found in conventional fermentation methods. Some organic acids, such as lactic, acetic, and propionic acids, and their salts, however, may be produced economically from fermentations of sugars (e.g., lactose, glucose, fructose, sucrose) and organic acids (e.g., lactate, pyruvate) present in culture media or biomass (e.g., whey, corn steep liquor, sulfite liquor).
Lactic acid is an important specialty chemical with a current market of about 40 million lbs per year in the U.S. It is currently used both as a food additive and as an industrial chemical. Lactic acid is produced either synthetically or biologically. The synthetic product is preferred in some industrial applications because of its high purity. However, fermentation can produce the pure L(+)- or D(-)-isomer or a mixture of the two, depending on the bacterium used. Such specific lactic acid isomers are important to the production of biodegradable lactic acid polymers which may replace polyesters and other non-biodegradable plastics in many applications. Thus, pure (polymer grade) lactic acid may become a commodity chemical in the near future. Commercial interests in lactic acid fermentation are high. Recently, several new lactic acid fermentation plants have been or are being constructed in the U.S., including two whey-based fermentation plants.
Propionic acid is an important chemical used in the production of cellulose plastics, herbicides, and perfumes. Propionic acid is also an important mold inhibitor. Its calcium, sodium, and potassium salts are widely used as food and feed preservatives. Presently, commercial production of propionic acid is predominantly by petrochemical routes. However, interests in producing propionic acid and calcium propionate from whey lactose and other cheap biomass using propionibacteria are high.
Acetic acid is an important mw material in the chemical industry. The production of acetic acid in the U.S. was .about.3.2 billion pounds in 1992. One major new use for acetic acid is in roadway deicing, where calcium magnesium acetate (CMA), produced from glacial acetic acid and dolomitic lime, is used as a deicer to replace road salt. Another similar new use for acetic acid is to use potassium acetate to replace urea and glycols in airport runways deicing. At the present time, commercial production of glacial acetic acid is exclusively by the petrochemical route. However, there has been high interest in producing acetic acid and acetate from fermentations of various biomass, including whey.
One of the incentives for producing inexpensive acetic acid or acetate is the interest in calcium magnesium acetate (CMA) for use as a substitute for road or highway deicing salt. Salt and chemical deicers continue to be the major way to control snow and ice on highways. From 10 to 14 million tons of road salt are used annually in the United States and Canada. Salt is an extremely effective snow and ice control agent and is very cheap. However, extensive use of rock salt (sodium chloride) as a deicing chemical has resulted in millions of dollars of loss each year due to its damage to highways and motor vehicles. Salt is extremely corrosive to concrete and metals, which are an integral part of the nation's infrastructure. Salt also is harmful to vegetation and poses an environmental threat to groundwater quality in some regions. A recent study in New York State showed that while a ton of road salt costs only $30, it causes more than $1,400 in damage. The Federal Highway Administration has long recognized this problem and recently has identified calcium magnesium acetate (CMA) as one of the most promising alternative road deicers.
CMA is a mixture of calcium acetate and magnesium acetate, currently being manufactured by reacting glacial acetic acid with dolomitic lime (Ca/MgO) or limestone (Ca/MgCO.sub.3). CMA has a deicing ability comparable to salt. In contrast to salt, CMA is noncorrosive to vehicles, not harmful to highway concrete, bridges and vegetation, and has no identified environmental concerns. However, the present cost for CMA is high--$650/ton versus $30/ton for salt. This makes it too expensive to use CMA even though all of the material cost due to CMA can be offset by the savings in other costs. For this reason, CMA is currently used only in limited areas where corrosion control is required and in environmentally sensitive areas to protect vegetation and ground water from salt poisoning. The use of CMA as a chemical deicer, however, will be widely accepted if the CMA production cost can be reduced to $300/ton ($0.15/lb) or less. About 75% of the production costs for the present commercial CMA deicer can be attributed to the glacial acetic acid, which costs at about $0.2/lb, used in its manufacturing. A low-cost CMA may be produced from whey lactose using the new device and method disclosed in this invention.
A new method has now been devised wherein sugar containing solutions, such as whey or its equivalent lactose containing solution, may be fermented with cells such as anaerobic homolactic and homoacetic bacteria in the new immobilized cell bioreactors to achieve a broth containing the desired products, such as acetic acid and acetate. The organic acids present in the broth may then be recovered and concentrated to form organic salts by using the two-step extraction method.
It is therefore the object of the invention to provide an effective apparatus and method for convening organic materials into more useful organic materials through fermentations.
It is also an object of the present invention to provide an improved way for converting sugars or sugar-containing biomass into organic acids, the salts of such acids and other biochemicals through the use of microorganisms.
A further object of the present invention is to provide a method and apparatus for converting lactose into useful organic acids or organic salts.
It is a further object of the invention to provide an apparatus and method for converting whey lactose (or its sugar or lactose containing equivalent) into useful organic acids or organic
It is a further object of the invention to provide a method and apparatus for continuously converting whey lactose (or its sugar or lactose containing equivalent) into useful organic acids or organic salts.
Another object of the present invention is to provide a new, improved, immobilized cell bioreactor for uses in various bioprocesses, including fermentations, biotransformations, and biofiltrations, for the purpose of converting organic materials into something more desirable.